Analysis of small inorganic cations of ammonium, alkali and alkaline-earth metal ions is commonly required for various purposes, such as control of food quality, clinical diagnosis and environmental monitoring. Analysis of small molecules and ions by capillary electrophoresis (CE) is of growing interest because of its simplicity, selectivity and low operation cost (P. Jandik and G. K. Bonn, Capillary Electrophoresis of Small Molecules and Ions, VCH Publishers, New York, 1993).
Studies on analysis of the small inorganic cations by CE focus primarily on finding solutions to low selectivity resulting from insufficient differences in mobilities of the hydrated cations and poor detectability due to lack of chromophores (F. Foret, S. Fanali, A. Nardi and P. Bocek, Capillary Zone Electrophoresis of Rare Earth Metals with Indirect UV Absorbance Detection, Electrophoresis, 11(1990) 780-783; L. Gross and E. S. Yeung, Indirect Fluorometric Detection of Cations in Capillary Zone Electrophoresis, Anal. Chem., 62 (1990) 427-431; A. Weston, P. R. Brown, P. Jandik, W. R. Jones and A. L. Heckenberg, Factors Affecting the Separation of Inorganic Metal Cations by Capillary Electrophoresis, J. Chromotogr. 593 (1992) 289-295).
As complexes have usually different charge to mass ratio from those of the hydrated cations, effective mobilities of the cations can be manipulated to enhance selectivity in CE separation of the small inorganic cations by introducing proper complexing agents in CE buffer. The complexing agents mainly includes multidentate complexing agents of donor atoms of oxygen. To differentiate ammonium from potassium ions, addition of a crown ether to buffer is effective.
A common and simple solution to poor detectability of the small cations is to provide chromophore co-ions of strong UV/visible absorption (or fluorescence) in electrophoresis buffer and to make use of indirect optical detection. The no-absorbing cations displace the chromophore co-ions while the cations are driven to move in electrophoresis buffer by electrophoretic force and electroosmotic flow (EOF). The cations are detected by decrease of absorbance. Chromophore co-ions for separation and detection of the small cations are usually nitrogen-containing heterocyclic compounds and substitutes of benzylamines.
Other variables are also known to affect selectivity and detectability in the separation of the small inorganic cations by CE. Variables related to buffer are pH, pH buffering capacity, ionic strength and conductivity, mobilities and pK.sub.a 's of co-ions, wavelengths of absorption maxima and molar absorption coefficients of co-ions, concentrations of complexing agents and co-ions, etc. Variables related to capillary electrophoresis system are temperature, voltage, capillary dimensions, etc. Some variables are independent ones. Some are not. Development of buffer used for the analysis of the small inorganic cations with high selectivity and detectability needs the overall optimization of the variables above.
The analysis of the small inorganic cations of ammonium ions, alkali and alkaline-earth metal ions by CE with indirect optical detection has been an object of several research papers and patents. Beck and Engelhardt proposed the imidazolium cation as a chromophore co-ion of electrophoresis buffer for the separation of alkali and alkaline-earth metal cations (W. Beck and H. Engelhard, Capillary electrophoresis of organic and inorganic cations with indirect UV detection, Chromatographia, 33(7/8) (1992) 314-316). Jones et al. documented 4-methylbenzylamine as a chromophore co-ion and a group of complexing agents (citrate, succinate, tartrate, hydroxyisobutyrate, and oxalate) for the separation of cations in their patents (W. R. Jones, P. Jandik, M. Merion and A. Weston, Method for separating ionic species using capillary electrophoresis, European Patent 0442315 A1; W. R. Jones and P. Jandik, Method for separating ionic species using capillary electrophoresis, U.S. Pat. No. 5,156,724). Shi and Fritz compared different chromophores (phenylethylamine, benzyl amine, p-toluidine and 4-methylbenzyl amine) and complexing agents (hydroxyisobutyric, phthalic, malonic, tartaric, lactic and succinic acids) for the separation of alkali, alkaline-earth and other metal ions (Y. Shi and J. S. Fritz, Separation of metal ions by capillary elecrtrophoresis with a complexing electrolyte, J. Chromatogr., 640 (1993) 473-479). Lin et al. studied the role of complexing agents (acetic, glycolic, lactic, hydroxyisobutyric, oxalic, malonic, tartaric, and succinic acids) added to imidazole-based electrophoresis buffer in the separation of alkali and alkaline-earth metal cations (T. I. Lin et al, Y. H. Lee and Y. C. Chen, Capillary electrophoretic analysis of inorganic cations-Role of complexing agent and buffer pH, J. Chromatogr. A, 654 (1993) 167-176). In another study, Lee and Lin studied effects of chromophore co-ions (imidazole, benzylamine, ephedrine and pyridine) and complexing agents (glycolic, hydroxyisobutyric and succinic acids) (Y. H. Lee and T. I. Lin, Determination of metal cations by capillary electrophoresis -Effect of background carrier and complexing agents, J. Chromatogr. A. 675 (1994) 227-236). Francois et al. made a close study on effect of the concentrations of lactic acid and 18-crown-6 in electrophoresis buffer upon the separation of ammonium, alkali and alkali-earth cations by CE (C. Francois, Ph. Morin and M. Dreux, Effect of the concentration of 18-crown-6 added to the electrolyte upon the separation of ammonium, alkali and alkaline-earth cations by capillary electrophoresis, J. Chromatogr. A. 706 (1995) 535-553; C. Francois, Ph. Morin and M. Dreux, Separation of transition metal cations by capillary electrophoresis-Optimization of complexing agent concentrations (lactic acid and 18-crown-6) J. Chromatogr. A. 717 (1995) 393-408). Shi and Fritz, and Yang et al. studied on effect of aqueous-organic media on the separation of ammonium and metal ions (Y. Shi and J. S. Fritz, New electrolyte systems for the determination of metal cations by capillary zone electrophoresis, J. Chromatogr. A. 671 (1994) 429-435; Q. Yang, J. Smeyers-Verbeke, W. Wu, M. S. Khots and D. L. Massart, Simultaneous separation of ammonium and alkali, alkaline earth and transition metal ions in aqueous-organic media by capillary ion analysis, J. Chromatogr. A. 688 (1994) 339-349). All the above mentioned electrophoresis buffer systems contained multidentate complexing agents of donor atoms of only oxygen. Wang and Li studied migration behavior of alkali and alkaline-earth metal ion-EDTA complexes (T. Wang and S.F.Y. Li, Migration behavior of alkali and alkaline-earth metal ion-EDTA complexes and quantitative analysis of magnesium in real samples by capillary electrophoresis with indirect ultraviolet detection, J. Chromatogr. A. 707 (1995) 343-353). EDTA is a widely used multidentate complexing agent of donor atoms of oxygen and nitrogen. The results showed that EDTA-pyridine buffer system was not suitable to separate and determine calcium due to very high stability of EDTA-Ca complex. None of the published CE methods using available buffer systems has demonstrated the capability of separating all eight of the common small inorganic cations (NH.sub.4.sup.+, Li.sup.+, Na.sup.+, K.sup.+, Mg.sup.2+, Ca.sup.2+, Sr.sup.2+ and Ba.sup.2+) with satisfactory resolution for the purposes of qualitatively screening or quantitatively determining the cations in real samples, especially for samples of complex matrices and high variation of concentrations of the cations. Therefore, there is a need for invention of CE buffer to be used for analyzing the small inorganic cations in real samples with higher selectivity and resolution than the available buffer systems.